Vpliv plazmida na uspešnost regeneracije in izražanja gfp gena v tobaku (Nicotiana tabacum L.)

Niko PAVLIN, Zlata LUTHAR

Povzetek


Listne izsečke tobaka smo transformirali z bakterijo Agrobacterium tumefaciens (A. t.) in plazmidom pBIN mgfp5-ER, ki ima eno kopijo zeleno fluorescentnega gfp gena in A. t.-pART27 2mgfp5-ER, ki ima dve kopiji gfp gena. Oba plazmida imata vgrajen še selekcijski nptII gen za odpornost na antibiotik kanamicin. Prisotnost zeleno fluorescentnega mGFP-ER proteina smo z epifluorescentnim mikroskopom zasledili v posameznih celicah 3 dni po transformaciji z A. t.-pART27 2mgfp5-ER in po 6 dneh tudi v celicah transformiranih z A. t.-pBIN mgfp5-ER. Regeneracija je bila po okužbi A. t.-pART27 2mgfp5-ER v večini primerov direktna, brez vmesne faze kalusa in hitrejša, saj so prve globularne strukture nastale že 10–12 dni po transformaciji ter dosežena je bila 204 % regeneracija. Prve globularne strukture po okužbi z A. t.-pBIN mgfp5-ER so se pojavljale šele po 18 dneh, nastalo je več kalusa in regeneracija je bila nižja, samo78,4 %. Pri vseh 149 nastalih regenerantih smo z dupleks PCR analizo potrdili prisotnost fragmentov dolžine 650 bp, značilnih za selekcijski nptII gen in fragmentov dolžine 422 bp, značilnih za markerski gfp gen.

Ključne besede


Nicotiana tabacum; markerski gfp gen; selekcijski nptII gen; uspešnost transformacije; izražanje transgenov; DNA analiza

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Literatura


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DOI: http://dx.doi.org/10.14720/aas.2014.103.1.09

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